Netrins, a family of secreted proteins, are not only required for axon guidance and neuronal migration during development, but are also involved in angiogenesis and tumorigenesis. Recent studies including ours indicate that DSCAM (Down syndrome cell adhesion molecule), a transmembrane protein of the immunoglobulin superfamily, functions as a new netrin receptor required for spinal cord commissural axon outgrowth and pathfinding. This application proposes to investigate the molecular mechanisms mediating neuronal responses to netrin-1 with a focus on studying the coordination of signal transduction cascades downstream of different netrin receptors, for instance, DCC, DSCAM and UNC5. Aim 1. Determine the coordinating role of JNK1 in netrin signaling. Our preliminary results suggest that JNK1 is involved in netrin signaling. To further characterize the coordinating role of JNK1 in DCC and DSCAM downstream signaling, we plan to examine the regulation of JNK1 activity by netrin-1 in HEK293 cells and primary neurons, as well as the functional importance of JNK1 in netrin-induced axon outgrowth and attraction. Aim 2. Untangle the coordination of protein phosphorylation in netrin signaling. Protein phosphorylation plays a critical role in netrin signaling during embryonic development. To study the coordination of signal transduction cascades downstream of DSCAM and DCC, we will examine the regulation of protein phosphorylation in netrin/DSCAM signaling, the coordination of protein phosphorylation in netrin/DSCAM and netrin/DCC signaling, relationship of Src family kinases and JNK1 in netrin signal transduction cascades and the functional coordination of DSCAM and DCC in netrin-induced axon outgrowth and attraction. Aim 3. Investigate the coordination of DSCAM and UNC5 in netrin-mediated repulsion. Netrin-1 can act as an attractant or repellent in axon guidance and neuronal migration, depending on when it binds to UNC5. DSCAM forms a receptor complex with UNC5C in HEK293 cells and primary neurons, indicating that they may coordinate in netrin-mediated neuronal repulsion. We plan to examine the distribution and co-localization of DSCAM and UNC5C in the developing nervous system, the coordination of netrin/DSCAM and netrin/UNC5C downstream signaling and the functional role of DSCAM in netrin-mediating axon repulsion. These experiments will shed new light on the coordination of netrin downstream signal transduction cascades and help us better understand how the brain establishes its complex and precise wiring during development. PUBLIC HEALTH RELEVANCE: Coordination of signal transduction cascades downstream of guidance cues is crucial for neurons to find their targets and establish neural circuitry in the developing nervous system. The secreted proteins netrins are prototypical guidance cues for projecting axons and migrating neurons. Studying signal transduction of netrins will not only help us understand how the brain establishes its complex and precise wiring during development, but also provide a basis to design new strategies for injury-related diseases, such as stroke and spinal cord injuries.